[Show abstract][Hide abstract] ABSTRACT: This study is devoted to the antiviral activity of peptide fragments from the PB1 protein – a component of the influenza A RNA polymerase. The antiviral activity of the peptides synthesized was studied in MDCK cell cultures against the pandemic influenza strain A/California/07/2009 (H1N1) pdm09. We found that peptide fragments 6-13, 6-14, 26-30, 395-400, and 531-540 of the PB1 protein were capable of suppressing viral replication in cell culture. Terminal modifications i.e. N-acetylation and C-amidation increased the antiviral properties of the peptides significantly. Peptide PB1 (6 – 14) with both termini modified showed maximum antiviral activity, its inhibitory activity manifesting itself during the early stages of viral replication. It was also shown that the fluorescent-labeled analog of this peptide was able to penetrate into the cell. The broad range of virus-inhibiting activity of PB1 (6-14) peptide was confirmed using a panel of influenza A viruses of H1, H3 and H5 subtypes including those resistant to oseltamivir, the leading drug in anti-influenza therapy. Thus, short peptide fragments of the PB1 protein could serve as leads for future development of influenza prevention and/or treatment agents.
Antiviral Research 01/2015; 113:4-10. · 3.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Influenza virus is serious human pathogen leading to high morbidity and mortality all over the world. Due to high rate of mutation, it is able to fast development of drug resistance that makes necessary to search novel antivirals with broad range and alternative targets. In the present study we describe synthesis and anti-viral activity of novel derivatives of usnic acid (2,6-diacetyl-7,9-dihydroxy-8,9b-dimethyl-1,3(2H,9bH)-dibenzo-furandione). It is shown that anti-viral activity of usnic acid can be increased by side moieties introduction. The modification with chalcones appeared to be the most effective. Our study revealed that (−)-usnic acid exhibited higher antiviral activity than its (+)-enantiomer, but in the pairs of enantiomer derivatives such as enamines, pyrazoles and chalcones, the (+)-enantiomers were more potent inhibitors of the virus. For other groups of compounds the inhibiting activities of the enantiomers were comparable. Further optimization of the structure could therefore result in development of novel anti-influenza compound with alternative target and mechanism of virus-inhibiting action.
[Show abstract][Hide abstract] ABSTRACT: Influenza A virus is one of the major human pathogens. Despite numerous efforts to produce absolutely effective anti-influenza drugs or vaccines, no such agent has been developed yet. One of the main reasons for this complication is the high mutation rate and the specific structure of influenza A viruses genome. For more than 25 years since the first mapping of the viral genome, it was believed that its 8 genome segments encode 10 proteins. However, the proteome of influenza A viruses has turned out to be much more complex than previously thought. In 2001, the first accessory protein, PB1-F2, translated from the alternative open reading frame, was discovered. Subsequently, six more proteins, PB1-N40, PA-X, PA-N155, PA-N182, M42, and NS3, have been found. It is important to pay close attention to these novel proteins in order to evaluate their role in the pathogenesis of influenza, especially in the case of outbreaks of human infections with new avian viruses, such as H5N1 or H7N9. In this review we summarize the data on the molecular mechanisms used by influenza A viruses to expand their proteome and on the possible functions of the recently discovered viral proteins
[Show abstract][Hide abstract] ABSTRACT: Recombinant cold-adapted strain A/HK/Otar/6:2/2010 (H3N8) constructed by the method of classic genetic reassortment, inherited the hemagglutinin and neuraminidase genes from the epizootic A/equine/Otar/764/2007 (Н3N8) virus, and genes of non-glycosylated proteins from attenuation donor A/Hong Kong/1/68/162/35 (H3N2). The resulted virus was of cold-adapted (efficient growth at 25°C) and temperature-sensitive (restricted replication at 39°C) phenotype. Like the parental virus of subtype H3N2 the resulted reassortant was attenuated, with limited replication in lungs of mice (1.75 lgEID50/ml) versus replication in turbinate (2.5 lgEID50/ml). Intranasal immunization of mice with A/HK/Otar/6:2/2010 (H3N8) reassortant did not induce animal weight reduction in contrast to epizootic A/equine/Otar/764/2007 (Н3N8) virus.
Journal of Equine Veterinary Science 06/2014; · 0.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Influenza is a continuing world-wide public health problem that causes significant morbidity and mortality during seasonal epidemics and sporadic pandemics. The purpose of the study was synthesis and investigation of antiviral activity of camphor-based symmetric diimines and diamines. A set of C2-symmetric nitrogen-containing camphor derivatives have been synthesized. The antiviral activity of these compounds was studied against rimantadine- and amantadine-resistant influenza virus A/California/7/09 (H1N1)pdm09 in MDCK cells. The highest efficacy in virus inhibiting was shown for compounds 2a-e with cage moieties bound by aliphatic linkers. The therapeutic index (selectivity index) for 2b exceeded that for reference compounds amantadine, deitiforin and rimantadine almost 10-fold. As shown by structure-activity analysis, the length of the linker has a dramatic effect on the toxicity of compounds. Compound 2e with -C12H24- linker exhibited the lowest toxicity (CTD50=2216μM). Derivatives of camphor, therefore, can be considered as prospective antiinfluenza compounds active against influenza viruses resistant to adamantane-based drugs.
[Show abstract][Hide abstract] ABSTRACT: Conjugates of glycyrrhetic acid (GLA) with amino acids (L-isoleucine, -leucine, -valine, and -phenylalanine) were synthesized by the acid-chloride method using methyl or tert-butyl esters of the acids. Tests in MDCK cell culture showed that the GLA conjugate with phenylalanine exhibited high antiviral activity against influenza A/H1N1/pdm09 virus (ED 50 =4.4 Pg/mL, SI = 161)., influenza A/H1N1 virus, antiviral activity. Chemical modification of plant triterpenoids has in the last decade produced a whole series of lead compounds that are medically promising as new antitumor, antiviral, and antidiabetic agents. The pentacyclic triterpenoid glycyrrhetic acid (GLA, 1) is the product from acid and enzymatic hydrolysis of glycyrrhizic acid, the principal constituent of Glycyrrhiza glabra L. and G. uralensis Fisher roots, and is a promising natural product for creating new drugs to treat and prevent oncological and inflammatory diseases, hepatoprotectors, antioxidants, antiulcer and antiviral agents, etc. . The potential to produce new biologically active compounds through chemical modification of GLA has not been fully explored [2–5]. In continuation of our research on the synthesis of new biologically active GLA derivatives, we synthesized amino-acid conjugates using GLA 3-O-acetate (2) as starting material. It was converted into acid chloride 3 via reaction with thionylchloride (SOCl 2) in benzene (Scheme 1). The amino acids were methyl or tert-butyl (t-Bu) esters of L-amino acid hydrochlorides and were acylated via reaction with 3 in CH 2 Cl 2 in the presence of Et 3 N or N-methylmorpholine (NMM) to afford conjugates 4–8 in 60–70% yields. The yields of the protected conjugates were higher if the amino-acid t-Bu esters were used as the amines. Analytically pure 4 and 5 were isolated by column chromatography (CC) over silica gel (SG). Conjugates 6–8 were reprecipitated from aqueous EtOH. The protecting groups were removed without further purification. Conjugates 4 and 5 in dioxane:MeOH were treated with aqueous NaOH (4 N) and acidified subsequently with HCl solution (5%) in order to remove the 3-O-acetyl and methyl ester. The t-Bu ester protecting group of 6–8 was removed by trifluoroacetic acid (TFA) in CH 2 Cl 2 at 20–22°C. Then, the 3-O-acetyl protection was removed in dioxane by alkaline hydrolysis using aqueous NaOH (4 N). The resulting compounds were chromatographed over SG to afford free GLA conjugates 9–12 containing free amino acids L-isoleucine (Ile), -leucine (Leu), -valine (Val), and -phenylalanine (Phe) in 53–57% yields.
[Show abstract][Hide abstract] ABSTRACT: Live attenuated influenza vaccines (LAIVs) are being developed to protect humans against future epidemics and pandemics. This study describes the results of a double-blinded randomized placebo-controlled phase I clinical trial of cold-adapted and temperature sensitive H7N3 live attenuated influenza vaccine candidate in healthy seronegative adults.
The goal of the study was to evaluate the safety, tolerability, immunogenicity and potential shedding and transmission of H7N3 LAIV against H7 avian influenza virus of pandemic potential.
Two doses of H7N3 LAIV or placebo were administered to 40 randomly divided subjects (30 received vaccine and 10 placebo). The presence of influenza A virus RNA in nasal swabs was detected in 60.0% and 51.7% of subjects after the first and second vaccination, respectively. In addition, vaccine virus was not detected among placebo recipients demonstrating the absence of person-to-person transmission. The H7N3 live attenuated influenza vaccine demonstrated a good safety profile and was well tolerated. The two-dose immunization resulted in measurable serum and local antibody production and in generation of antigen-specific CD4(+) and CD8(+) memory T cells. Composite analysis of the immune response which included hemagglutinin inhibition assay, microneutralization tests, and measures of IgG and IgA and virus-specific T cells showed that the majority (86.2%) of vaccine recipients developed serum and/or local antibodies responses and generated CD4(+) and CD8(+) memory T cells.
The H7N3 LAIV was safe and well tolerated, immunogenic in healthy seronegative adults and elicited production of antibodies broadly reactive against the newly emerged H7N9 avian influenza virus.
PLoS ONE 01/2014; 9(2):e87962. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Development of safe container materials for targeted and controlled drug delivery to the right site in the body is one of the most important aspects of modern biotechnologies. In the last decade, a significant progress has been achieved in the study of nanostructured drug carriers, but the use of many nanomaterials is
fraught with the enormous risk because of their high toxicity. The real breakthrough became the use of porous silicon, which has such important properties as biocompatibility, bioavailability, and biodegradability, which makes it possible to use it for solving a wide range of biological and medical problems in the field of diagnosis and treatment of diseases, implantology, and biomolecular screening.
[Show abstract][Hide abstract] ABSTRACT: Currently, application of new technological solutions for the implementation of strategies for improving influenza vaccines are up to date and modern. In this study, we present data obtained in the evaluation of the safety and immunogenicity of the new Russian (national) split seasonal inactivated influenza vaccine ULTRIKS in comparison with a commercial vaccine VAXIGRIP in various age groups.
[Show abstract][Hide abstract] ABSTRACT: The development of influenza virus vectors with long insertions of foreign sequences remains difficult due to a small size and instable nature of the virus. Here, we used the influenza virus inherent property of self-optimization to generate a vector stably expressing long transgenes from the NS1 open reading frame (NS1-ORF). This was achieved by continuous selection of bright fluorescent plaques of a green fluorescence protein (GFP) expressing vector during multiple passages in mouse B16f1 cells. The newly generated vector acquired stability in interferon (IFN) competent cell lines and in vivo in murine lungs. Although, improved vector fitness was associated with the appearance of 4 coding mutations in PB2, HA and NS segments, the stability of the transgene expression was primarily dependent on the single mutation Q20R in the nuclear export protein (NEP). Importantly, a longer insert, such as a cassette of 1299 nts encoding two Mycobacterium tuberculosis Esat6 and Ag85A proteins, could substitute the GFP-transgene. Thus, the inherent property of the influenza virus to adapt can also be used to adjust a vector backbone to a stable expression of long transgenes.
Journal of General Virology 11/2013; · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Autophagy plays an important role in neoplastic transformation of cells and in resistance of cancer cells to radio- and chemotherapy. p62 (SQSTM1) is a key component of autophagic machinery which is also involved in signal transduction. Although recent empirical observations demonstrated that p62 is overexpressed in variety of human tumors, a mechanism of p62 overexpression is not known. Here we report that the transformation of normal human mammary epithelial cells with diverse oncogenes (RAS, PIK3CA and Her2) causes marked accumulation of p62. Based on this result, we hypothesized that p62 may be a feasible candidate to be an anti-cancer DNA vaccine. Here we performed a preclinical study of a novel DNA vaccine encoding p62. Intramuscularly administered p62-encoding plasmid induced anti-p62 antibodies and exhibited strong antitumor activity in four models of allogeneic mouse tumors - B16 melanoma, Lewis lung carcinoma (LLC), S37 sarcoma, and Ca755 breast carcinoma. In mice challenged with Ca755 cells, p62 treatment had dual effect: inhibited tumor growth in some mice and prolonged life in those mice which developed tumor size similar to control. P62-encoding plasmid has demonstrated its potency both as a preventive and therapeutic vaccine. Importantly, p62 vaccination drastically suppressed metastasis formation: in B16 melanoma where tumor cells where injected intravenously, and in LLC and S37 sarcoma with spontaneous metastasis. Overall, we conclude that a p62-encoding vector(s) constitute(s) a novel, effective broad-spectrum antitumor and anti-metastatic vaccine feasible for further development and clinical trials.
[Show abstract][Hide abstract] ABSTRACT: The synthesis and biological evaluation of a novel series of dimeric camphor derivatives are described. The resulting compounds were studied for their antiviral activity, cyto- and genotoxicity. Compounds 3a and 3d in which the quaternary nitrogen atoms are separated by the C5H10 and С9H18 aliphatic chain, exhibited the highest efficiency as an agent inhibiting the reproduction of the influenza virus A(H1N1)pdm09. The cytotoxicity data of compounds 3 and 4 revealed their moderate activity against malignant cell lines; compound 3f had the highest activity for the CEM-13 cells. These results show close agreement with the data of independent studies on toxicity of these compounds, in particular that the toxicity of compounds strongly depends on spacer length.
[Show abstract][Hide abstract] ABSTRACT: In this study, we assessed the immunogenicity and safety of one dose (7.5 or 15 μg of hemagglutinin, HA) of a whole virion inactivated pre-pandemic influenza vaccine adjuvanted with aluminum hydroxide in humans. The vaccine strain was made by reverse genetics from the highly pathogenic avian A/chicken/Astana/6/05 (H5N1) clade 2.2 strain isolated from a dead bird in Kazakhstan. The humoral immune response was evaluated after a single vaccination by hemagglutination inhibition (HI) and micro neutralization (MN) assays. The vaccine was safe and immunogenic, inducing seroconversion in 55% of the evaluated patients with a geometric mean titer (GMT) of 17.1 and a geometric mean increase (GMI) of 3.42 in a dose of 7.5 μg in HI test against the vaccine strain. The rate of seroconversion increased up to 70% when the dose of 15 μg was used. A percentage of individuals achieving anti-HA titer ≥1:40 was 52.5% and 57.5% for the 7.5 and 15 μg dose groups, respectively. Similar results were obtained when antibodies were analyzed by an MN test. Substantial cross-neutralization titers (seroconversion in 35% and 52.5% of subjects in both dose groups, respectively) were detected against a heterologous clade 1 strain NIBRG14 (H5N1). Thus, one dose of this whole virion pre-pandemic vaccine adjuvanted with aluminum has the potential to be effective against H5N1 viruses of different clades.
Clinical and vaccine Immunology: CVI 06/2013; · 2.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Polyhydroxyfullerenes, fullerenols with various contents of hydroxy groups, C60(OH)12 – 14, C60(OH)18 – 24, and C60(OH)30 – 38, were synthesized. The first group was insoluble in water and exhibited no biological activity when introduced into cell cultures as suspensions. The other two versions of fullerenols possessed a broad spectrum of antiviral activity in vitro against actual strains of human influenza virus A(H1N1) and A(H3N2), avian influenza A(H5N1), human herpes simplex virus, adenovirus, and respiratory-syncytial virus. The water-soluble versions of fullerenol were non-toxic in vitro toward human and animal cells of various tissue origins. Besides the antiviral activity, the fullerenols demonstrated a protective effect against UVA-induced phototoxicity. The fullerenol version C60(OH)18 – 24 had the maximum biological activity for all studied parameters. The soluble biologically active versions of fullerenol could find application in pharmacology because they could serve as a basis for new effective and non-toxic antiviral and cytoprotective drugs.
[Show abstract][Hide abstract] ABSTRACT: A new approach for super-expression of the influenza virus epitope M2e in plants has been developed on the basis of a recombinant Tobacco mosaic virus (TMV, strain U1) genome designed for Agrobacterium-mediated delivery into the plant cell nucleus. The TMV coat protein (CP) served as a carrier and three versions of the M2e sequence were inserted into the surface loop between amino acid residues 155 and 156. Cysteine residues in the heterologous peptide were thought likely to impede efficient assembly of chimeric particles. Therefore, viral vectors TMV-M2e-ala and TMV-M2e-ser were constructed in which cysteine codons 17 and 19 of the M2e epitope were substituted by codons for serine or alanine. Agroinfiltration experiments showed that the chimeric viruses were capable of systemically infecting Nicotiana benthamiana plants. Antisera raised against TMV-M2e-ala virions appear to contain far more antibodies specific to influenza virus M2e than those specific to TMV carrier particle (ratio 5:1). Immunogold electron microscopy showed that the М2е-epitopes were uniformly distributed and tightly packed on the surface of the chimeric TMV virions. Apparently, the majority of the TMV CP-specific epitopes in the chimeric TMV-M2e particles are hidden from the immune system by the M2e epitopes exposed on the particle surface. The profile of IgG subclasses after immunization of mice with TMV-M2e-ser and TMV-M2e-ala was evaluated. Immunization with TMV-M2e-ala induced a significant difference between the levels of IgG1 and IgG2a (IgG1/IgG2a=3.2). Mice immunized with the chimeric viruses were resistant to five lethal doses (LD50) of the homologous influenza virus strain, A/PR/8/34 (H1N1) and TMV-M2e-ala also gave partial protection (5LD50, 70% of survival rate) against a heterologous strain influenza A/California/07/2009 (H1N1) (4 amino acid changes in M2e). These results indicate that a new generation candidate universal nanovaccine against influenza based on a recombinant TMV construct has been obtained.
Current pharmaceutical design 02/2013; · 4.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Advances in transient expression technologies have allowed the production of milligram quantities of proteins within a matter of days using only small amounts (tens of grams) of plant tissue. Among the proteins that have been produced using this approach are the structural proteins of viruses which are capable of forming virus-like particles (VLPs). As such particulate structures are potent stimulators of the immune system, they are excellent vaccine candidates both in their own right and as carriers of additional immunogenic sequences. VLPs of varying complexity derived from a variety of animal viruses have been successfully transiently expressed in plants and their immunological properties assessed. Generally, the plant-produced VLPs were found to have the expected antigenicity and immunogenicity. In several cases, including an M2e-based influenza vaccine candidate, the plant-expressed VLPs have been shown to be capable of stimulating protective immunity. These findings raise the prospect that low-cost plant-produced vaccines could be developed for both veterinary and human use.
Current pharmaceutical design 02/2013; · 4.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In January 2012, influenza virus researchers from around the world announced a voluntary pause of 60 days on any research involving highly pathogenic avian influenza H5N1 viruses leading to the generation of viruses that are more transmissible in mammals. Now that the aims of the voluntary moratorium have been met in some countries and are close to being met in others, we declare an end to the voluntary moratorium on avian flu transmission studies.
[Show abstract][Hide abstract] ABSTRACT: Background Recombinant hemagglutinin (rHA) and neurominidase (rNA) developed in our investigation are amino acid sequence consensus variants of H1N1 2009 subtype influenza virus strain, also including immunogenic epitopes typical for other influenza virus subtypes (H3N1 and H5N1). Substitutions were made: typical for Russian virus isolates (in HA - S220T, NA - D248N) and in active centers of molecules - R118L, R293L, R368L; C92S, C417S to increase recombinant proteins stability in E. coli. The aim of the present work was to study immunogenicity of the obtained rHA and rNA. Material and Methods Fragments aa 83-469 of NA and aa 61-287 of HA were chosen because they include the main B-cell epitopes and are the minimal structures for correct folding of target proteins. The designed nucleotide sequences were synthesized and purified and the expression of rNA and rNA were analyzed. For immunization and virus challenge we used influenza viruses A/California/04/2009 (H1N1), A/PR/8/34 (H1N1), A/Perth/16/2009 (H3N2), A/Chicken/Kurgan/05/2005 R.G. (H5N1), and B/Florida/04/2006. Specific IgG levels were determined by ELISA in 96-well ELISA plates. Significant differences of survival in mouse groups were analyzed by Mantel-Cox (log-rank) and Gehan-Breslow-Wilcoxon tests. Results The obtained results demonstrate the high immunogenicity and ability of indicated proteins mixture to provide similar cross-protection against influenza viruses of the H1N1 subtype. Conclusions The data obtained suggest efficient pluripotent vaccine creation based on HA and NA conservative regions.
Medical science monitor basic research. 01/2013; 19:221-7.
[Show abstract][Hide abstract] ABSTRACT: A mirror-symmetry motif was discovered in the N-terminus of the influenza virus PB1 protein. Structure of peptide comprised of the corresponding part of PB1 (amino acid residues 6-25) was investigated by circular dichroism and in silico modeling. We found that peptide PB1 (6-25) in solution assumes beta-hairpin conformation. A truncated peptide PB1 (6-13), containing only half of the mirror-symmetry motif, appeared to stabilize the beta-structure of the original peptide and, at high concentrations, was capable of reacting with peptide to form insoluble aggregates in vitro. Ability of PB1 (6-13) peptide to interact with the N-terminal domain of PB1 protein makes it a potential antiviral agent that inhibits PA-PB1 complex formation by affecting PB1 N-terminus structure.
International Journal of Peptides 01/2013; 2013:370832.